Approximately 70% of therapeutic proteins such as antibodies, growth factors, cytokines, hormones, and clotting factors are glycoproteins, which are proteins modified post-translationally by the attachment of glycans. Most recombinant therapeutic glycoproteins are produced in mammalian expression systems because the location, number, and structure of N-glycans have been shown to affect the bioactivity, solubility, stability, pharmacokinetics, immunogenicity, and clearance rate of therapeutic glycoproteins. Two differences in the protein glycosylation machinery of humans and other mammals account for differences in the glycosylation patterns of glycoproteins produced by human cells and glycoproteins produced by other mammalian cells such as rodent cells.
First, humans cannot synthesize a terminal galactose-alpha-1,3-galactose moiety (also known as alpha-Gal or α-Gal) on N-glycans. The enzyme glycoprotein alpha-1,3-galactosyltransferase (Ggta1) forms the α-Gal moiety by linking a galactose residue via an α-1,3 gycosidic bond to a terminal galactose of the N-glycan. Humans apparently have a GGTA1 gene, but it is an expressed pseudogene encoding a non-functional truncated protein containing the first four translated exons but missing the two catalytic exons. Even though humans do not express a functional Ggta1 enzyme and, therefore, do not synthesize α-Gal moieties many humans produce antibodies against this structure.
Second, humans cannot synthesize the sialic acid, N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is produced by the hydroxylation of CMP-N-acetylneuraminic acid (Neu5Ac) to CMP-Neu5Gc by the enzyme CMP-Neu5Ac hydroxylase (Cmah). Although the human CMAH gene is irreversibly mutated, preventing the expression of Neu5Ac, traces of Neu5Gc have been detected in human serum. It appears that nonhuman Neu5Gc can be metabolically incorporated into human tissues from certain mammalian-derived foods, such that essentially all humans have Neu5Gc-specific antibodies, sometimes at high levels.
Chinese Hamster Ovary (CHO) cells are widely used for the manufacture of protein therapeutics, in part because they were assumed to produce proteins with human-like glycosylation patterns. For example, it was generally accepted that CHO cells lack the biosynthetic machinery to produce glycoproteins with α-Gal moieties. Moreover, even though CHO cells express Cmah and produce proteins having Neu5Gc units, the ratio of Neu5Gc to Neu5Ac units can be reduced by modifying CHO cell culture conditions. Despite the general acceptance that CHO cells were unable to synthesize α-Gal moieties, the CHO ortholog of Ggta1 has recently been identified (Bosques et al. Nature Biotechnol., 2010, 28(11):1153-1156). Because of the potential for hypersensitive reactions to recombinant therapeutic glycoproteins, there is a need for CHO cell lines and other non-human mammalian cell lines that produce glycoproteins devoid of α-Gal and/or Neu5Gc residues.